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Synthesis and evaluation of antimicrobial potential of copper nanoparticle

against agriculturally important Phytopathogens

Article · October 2016

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International Journal of Biology Research

International Journal of Biology Research

ISSN: 2455-6548, Impact Factor: RJIF 5.22
www.biotechjournals.com
Volume 1; Issue 4; September 2016; Page No. 41-47

Synthesis and evaluation of antimicrobial potential of copper nanoparticle against agriculturally

important Phytopathogens
1
SS Shende, 2 ND Gaikwad, 3 SD Bansod
1, 2
Vilasrao Deshmukh College of Agricultural Biotechnology, Latur. Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani,
Maharashtra, India
3
Chaitanya Agrobiotech Company, MIDC, Amravati, Maharashtra, India

Abstract
Biosynthesis of stable copper nanoparticles was achieved using Ocimum sanctum leaf extract. These biosynthesized copper
nanoparticles were characterized by using UV-Vis spectroscopy showed absorption of copper nanoparticles at 345nm. FTIR
spectrum of copper nanoparticles suggests that copper nanoparticles are surrounded by different organic molecules such as
terpenoids, alcohols, ketones, esters, aldehydes and carboxylic acid. TEM analysis showed that the copper nanoparticles were rod,
cylindrical and elliptical. The average particle size of copper nanoparticles was 25 nm with total concentration 300.29
particles/frame, 13.53 particles/ml and -14.9 mV zeta potential value.
The maximum antibacterial activity of copper nanoparticles was found against Xanthomonas axonopodis pv. punicae (17.25±0.95)
while Rhizoctonia solani (10±0.81) showed minimum activity with 0.01 mg/ml and 0.03mg/ml MIC respectively. The maximum
antifungal activity of copper nanoparticles was found against Alternaria carthami (18.5±1.7) while the minimum activity was
found against Rhizopus stolonifer (10.5±0.5). The MIC of the copper nanoparticles against fungus was found to be 0.06 mg/ml for
Alternaria carthami, Aspergillus niger, Fusarium oxysporum f.sp udum and 0.03 mg/ml for Colletotrichum gloeosporioides,
Colletotrichum lindemuthianum, and Drechslera sorghicola, Fusarium oxysporum f.sp. carthami, Fusarium oxysporum f.sp.
ciceri, Macrophomina phaseolina, Rhizoctonia bataticola and Rhizopus stolonifer.
Thus, copper nanoparticles can be exploited in the field of agriculture in formulation of various biopesticides, insecticides and
ecologically feasible effective management strategy against harmful phytopathogens.

Keywords: Cu nanoparticle, Tulsi, Phytopathogen, UV-Vis, FTIR, TEM, Particle analyzer, Zeta sizer, Antimicrobial activity

Introduction Materials and Methods

Development of green nanotechnology is generating interest
of researchers toward ecofriendly biosynthesis of
nanoparticles. Among the recent advancement in agricultural
sciences, metallic nanomaterials play a significant role in
crop protection because of its unique physical and chemical
properties, huge surface to volume ratio, structural stability
and strong affinity to their target (Kumar et al., 2010).
Nanoparticles remain bound to the cell wall of pathogen and
cuases deformity de to high energy transfer leading to its
death. Nanotechnological application in plant pathology
targets specific agricultural problems in plant pathogen
interactions and provide new perceptions for crop protection.
Nanocomposites fulfil the two most important criteria in
disease management: efficacy with minimal ecological
impact and less toxicity on humans. Copper based fungicides
produce highly reactive hydroxyl radicles which can damage
lipids, proteins, DNA and other Biomolecules. It plays an
important role in disease prevention and treatment of large
variety of plants (Borkow and Gabbay, 2005). With the
growing demand of pesticide worldwide to control the
pathogens and pests, there is an urgent need to tackle the
excessive usage of pesticeide and fertilizers by finding
alternatives. The potential applications of nanomaterials in
crop protection helps in the development of efficient and
potential approaches for the management of plant pathogens.
Sample collection
The Ocimum sanctum leaves were collected from Vilasrao
Deshmukh College of Agricultural Biotechnology, Latur
(M.S.) campus. Clove and orange were purchased from local
market. Copper sulphate was purchased from Hi-media
laboratories Mumbai, India.
Preparation of Ocimum sanctum leaf Extract
The fresh leaves of tulsi were washed several times with
distilled water to remove dust. When the leaves got
completely dried, they were chopped into fine pieces.5gm
chopped leaves of tulsi were boiled with 50 ml distilled water
at 60oC for 1 hour. The extract obtained was filtered through
Whatman No.1 filter paper and finally brown extract was
collected for further experiment.
Synthesis of Copper Nanoparticles
For the synthesis of copper nanoparticles, 50 ml of tulsi
leaf/orange peel/clove extract was mixed with 50 ml aqueous
solution of 1mM copper sulphate (1:1 ratio of plant extract
and copper solution) and stirred continuously for 2 min at
30oC. Reduction takes place rapidly which is indicated by the
change in colour of the solution. The mixture was incubated at
room temperature overnight. Mixture was centrifuged at 3500
rpm for 10min to get copper nanoparticles. The nanoparticles
were washed and dried at room temperature.

41
International Journal of Biology Research
Antimicrobial activity of copper nanoparticles
The antimicrobial activity of the nanoparticles was tested
against a three bacterial pathogens viz., Rhizoctonia solani,
Xanthomonas axonopodis pv. Citri, Xanthomonas
axonopodis pv. Punicae, and eleven fungal pathogens viz.,
Alternaria carthami, Aspergillus niger, Colletotrichum
gloeosporioides, Colletotrichum lindemuthianum,
Drechslera sorghicola, Fusarium oxysporum f.sp. carthami,
Fusarium oxysporum f.sp. ciceri, Fusarium oxysporum f.sp
udum, Macrophomina phaseolina, Rhizoctonia bataticola
and Rhizopus stolonifer.
1x104 cfu/ml bacterial culture and 1x105 spore/ml fungal
spore suspension were spread on nutrient and potato dextrose
agar media respectively. The disc were soaked in copper
10 min 20 min 30 min
Fig 1: Change in coloration of plant extract at different time intervals.
Characterization of copper nanoparticles
The synthesized copper nanoparticles were characterized by
using UV-Vis spectroscopy, FTIR, TEM, Particle size
analyzer (LM-20) and Malvern zeta sizer.
UV-Vis spectrophotometer
Fig 2: UV-Visible absorption spectra of biosynthesized copper nanoparticles.
nanoparticle solution and placed on media. Similarly, copper
sulphate and plant extract solutions were soaked on disc to
measure the control activity. Zone of inhibition was observed
after 24 hours in bacterial and 3-4 days in fungal cultures.
Results and Discussion
Synthesis of copper nanoparticles
Green synthesis of copper nanoparticles was achieved in
aqueous solution using plant extract as reducing agent. When
plant extract was mixed with copper sulphate solution, the
colour of aqueous solution was changed immediately within
10 min, which turns dark brown within 24 hours (Fig 1)
indicated the formation of copper nanoparticles.
40 min 1 hr 8 hr 24 hr
The synthesized copper nanoparticles were characterized by
using UV-Vis spectrophotometer and showed maximum
absorbance (2.15) at 345nm by using tulsi leaf extract as
reducing agent (fig.1)
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International Journal of Biology Research

Fourier Transform Infra-Red Spectroscopy (FTIR)
analysis
The FTIR spectra of copper nanoparticles are shown in
figure-2. The following peak were observed in spectrum and
the band at 3407∙06 cm-1 is assigned to the O-H stretching of
H-bonded alcohol and phenols. The band at 2926∙89 cm-1
attributed to O-H stretching of carboxylic acids. The band at
1628∙67 cm-1 corresponds to the NO2 stretching of Nitro
compound and presence of amide I and II, which arises due
to the carbonyl stretch and -N-H- stretch vibration in the
100
95
amide linkages of the proteins. The band at 1423.14 cm-1 is
related to the C-C stretching of aromatic ring structure,
scissoring and bending of alkanes. The band at 1081.91 cm -1
is related to the C-O stretching of alcohol, ether, esters,
carboxylic acid. The peak at 601.86, 437.73, 437.91 cm -1
indicated fingerprint region is complicated by the large
number of different vibrations that occur here. FTIR spectrum
of copper nanoparticles suggested that copper nanoparticles
were surrounded by different organic molecules such as
terpenoids, alcohols, ketones, aldehydes and carboxylic acid.

473.91
2926.89

437.73
90

601.86
1423.14
Transmittance [%]
80 85

1081.91
1628.67
3407.06
75
70

3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

Fig 3: FTIR spectra of biosynthesized copper nanoparticles by Ocimum sanctum.

C:\Program Files\OPUS_65\2014-2015\EXTERNAL 2014-15\FTIR 184\8-4-15\POWDER COPPER NANOPARTICLE.0 POWDER COPPER NANOPARTICLE
08/04/2015 SAIF IIT Bombay

Transmission Electron Microscopy (TEM) analysis Page 1/1

The size and shape of green synthesized copper
nanoparticles were analyzed using TEM particles were rod,
cylindrical and elliptical with particle size of 25nm.Particles
were well dispersed which was confirmed by TEM studies.
Selected area electron diffraction (SAED) patterns of the
copper nanoparticles synthesized using tulsi leaf extract were
investigated using HRTEM. The corresponding SAED of
copper nanoparticle patterns sample showed spotty ring
patterns without any additional diffraction spots shown in (fig
4).

43
International Journal of Biology Research

Fig 4: TEM image of Biosynthesized copper nanoparticle (A), higher magnified TEM image of biosynthesized copper
nanoparticles (B, C), with selected area electron diffraction (D).

Particle size analyzer (LM-20)

The nanoparticles tracking analyzer measurements showed total concentration 300.29 particles/ frame and 13.53
average particle size of copper nanoparticles was 25 nm with particles/ ml (fig.4).

Particle Size / Concentration

Fig 5: Particle size spectra of biosynthesized copper nanoparticles.

Malvern zeta sizer

The zeta potential is related to the surface charge density and nanoparticles in suspension. The biosynthesized copper
high magnitudes of zeta potential denote stability of the nanoparticles had -14.9 mV zeta potential value (Fig. no. 5).

Fig 6: Zeta potential of copper nanoparticles by zeta size analyzer.

44
International Journal of Biology Research
Antimicrobial activity of copper nanoparticle
The antimicrobial effect of biologically synthesized copper
nanoparticles was analyzed on the basis of the zone of
inhibition. Copper nanoparticles exhibited strong
antimicrobial activity against plant bacterial pathogen such
as Rhizoctonia solani, Xanthomonas axonopodis pv. citri,
Xanthomonas axonopodis pv. punicae and plant fungal
pathogens, such as Alternaria carthami, Aspergillus niger,
Colletotrichum gloeosporioides, Colletotrichum
lindemuthianum, Drechslera sorghicola, Fusarium
oxysporum f.sp. carthami, Fusarium oxysporum f.sp. ciceri,
Fusarium oxysporum f.sp. udum, Macrophomina phaseolina,
Rhizoctonia bataticola, Rhizopus stolonifer (Fig 7). The
maximum activity of copper nanoparticles was found against
bacterial pathogen Xanthomonas axonopodis pv. punicae
(17.25±0.95) while the minimum activity was found against
Rhizoctonia solani (10±0.81). The MIC of the copper
nanoparticles against bacteria was found to be 0.01 mg/ml
Alternaria carthami Aspergillus niger
Colletotrichum lindemuthianum Drechslera sorghicola
Fusarium oxysporum f.sp. cicero
for Rhizoctonia solani and 0.03mg/ml for Xanthomonas
axonopodis pv. punicae and Xanthomonas axonopodis pv.
citri.
The maximum activity of copper nanoparticles was found
against fungal pathogen Alternaria carthami (18.5±1.7) while
the minimum activity was found against Rhizopus stolonifer
(10.5±0.5). The MIC of the copper nanoparticles against
fungus Alternaria carthami, Aspergillus niger, Fusarium
oxysporum f.sp udum was found to be 0.06 mg/ml and 0.03
mg/ml for Colletotrichum gloeosporioides, Colletotrichum
lindemuthianum, and Drechslera sorghicola, Fusarium
oxysporum f.sp. carthami, Fusarium oxysporum f.sp. ciceri,
Macrophomina phaseolina, Rhizoctonia bataticola and
Rhizopus stolonifer. Control activity of copper sulphate
and tulsi plant extract was also tested. Tulsi plant extract did
not shown any antimicrobial activity whereas copper sulphate
shown relatively less activity as compared to copper
nanoparticles.
Colletotrichum gloeosporioides
Fusarium oxysporum f.sp. carthami
Fusarium oxysporum f.sp. udum
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International Journal of Biology Research

Macrophomina phaseolina Rhizoctonia bataticola Rhizopus stolonifer

Rhizoctonia solani Xanthomonas axonopodis pv. Citri Xanthomonas axonopodis pv. Punicae
Fig 7: Antimicrobial activity of copper nanoparticle against plant pathogenic bacteria and fungi.

Table 1: Antimicrobial activity of copper nanoparticles. nanoparticles in this study will presumably useful in
S. Name of the Diameter of zone of MIC formulation of various biopesticides, insecticides and
No. Microorganism inhibition (mm) (mg/ml) ecologically feasible effective management strategy against
Bacteria harmful pathogenic microorganisms.
1. Rhizoctonia solani 10±0.81 0.01
Xanthomonas axonopodis Acknowledgement
2. 13.5±1.29 0.03 The authors are grateful to the Department of Biotechnology,
pv. Citri
Xanthomonas axonopodis Government of India, New Delhi, for providing financial
3. 17.25±0.95 0.03
pv. Punicae support for above study.
Fungi
1. Alternaria carthami 18.5±1.7 0.06 References
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